The invention relates to recombinant proteins, particularly to viral reverse transcriptase enzymes produced by recombinant DNA technology, and specifically relates to reverse transcriptase derived from Moloney Murine Leukemia virus (MMLV) that is expressed from recombinant DNA in a bacterial host cell and that includes multiple histidine residues.
Retroviruses are a group of viruses whose genetic material consists of single-stranded RNA. Following adsorption and entry of retroviral RNA into the host cell, the viral RNA is used as a template for synthesis of a complementary DNA (cDNA) strand. The cDNA is then made double-stranded through the action of an enzyme having DNA polymerase activity; this double-stranded DNA integrates into the host genome. The RNA-directed DNA polymerase activity responsible for the synthesis of cDNA from the viral RNA template is commonly called reverse transcriptase (xe2x80x9cRTxe2x80x9d).
A number of retroviruses have been implicated as the causative agents of various cancers, and other diseases. A retrovirus, human immunodeficiency virus-1 (HIV-1), is the causal agent of acquired immunodeficiency syndrome (AIDS). Also, reverse transcriptase enzymes have become important reagents in molecular biology because of their ability to make cDNA from almost any RNA template. Reverse transcriptase is commonly used to make nucleic acids for hybridization probes and to convert single-stranded RNA into a double-stranded DNA for subsequent cloning and expression.
Reverse transcriptases have been used as a component of transcription-based amplification systems. These systems amplify RNA and DNA target sequences up to 1-trillion fold and have been previously described in detail (see Burg et al., PCT Patent Application WO 89/01050 (1988) and U.S. Pat. No. 5,437,990; Gingeras et al., PCT Patent Application WO 88/10315 (1988); Gingeras et al., European Patent Application EPO 0373960 (1989); Davey and Malek, European Patent Application EPO 0329822 (1988); Malek and Davey, PCT Patent Application WO 91/02814 (1989); Davey et al., U.S. Pat. Nos. 5,409,818 and 5,554,517; Davey et al., U.S. Pat. No. 5,466,586; Malek et al., U.S. Pat. No. 5,130,238; Kacian et al., European Patent Application EPO 0408295 A2 (1990) and U.S. Pat. Nos. 5, 399,491, 5,480,784, 5,824,518, 5,888,779 and 5,554,516), the experimental details of which are hereby incorporated by reference herein.
Some transcription-based amplification methods are especially convenient because the amplification reactions are isothermal. These systems are particularly suited for diagnostic tests in clinical laboratories. For example, detection of pathogens causing infectious diseases and gene sequences associated with cancers or genetic diseases are important uses of such tests. Reverse transcriptases are also employed as an initial step in some protocols in which the polymerase chain reaction (PCR) amplifies an RNA target (see Malek et al., U.S. Pat. No. 5,130,238 (1992); and Mocharla et al., 1990, Gene 99:271-275). In RT-PCR procedures, the reverse transcriptase is used to make an initial cDNA copy of the RNA target, which is then amplified by successive rounds of DNA replication using PCR.
Retroviral reverse transcriptases have three enzymatic activities: RNA-directed DNA polymerase activity, DNA-directed DNA polymerase activity, and RNAse H activity (Verma I., 1977, Biochim. Biophys. Acta 473: 1-38). The latter activity specifically degrades RNA contained in an RNA:DNA duplex. RNA strand degradation in RNA:DNA intermediates by RNAse H is an important component of some transcription-based amplification systems. RNAse H-mediated degradation of RNA is distinguishable from unwanted degradation due to contaminating nucleases, which interferes with amplification.
A disadvantage of the transcription-based amplification systems is their sensitivity to even trace amounts of nucleases. Because a number of important diseases may yield samples containing very few target nucleic acid molecules, detection of small amounts of target is often crucial for an accurate and timely diagnosis. Indeed, target amplification methods are most valuable when the number of target molecules is low. With low levels of input target nucleic acids, unwanted degradation of RNA targets, or of RNA or DNA reaction intermediates, can lead to amplification failures and consequent inaccurate diagnosis. Ribonuclease contamination is also a problem in RT-PCR reactions, because RNA target loss can result in amplification failure.
Ribonucleases are relatively ubiquitous and occur in high concentrations in a variety of biological materials, including in retrovirus preparations and cells commonly used to express recombinant proteins. Ribonucleases (xe2x80x9cRNasesxe2x80x9d) frequently contaminate RT preparations from a variety of sources and can interfere with cDNA synthesis, probe preparation and other uses besides target amplification. Often, an RNase inhibitor is added to a reaction mixture to minimize the deleterious effects of this contamination (e.g., see Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), pp. 8.11-8.13).
Commonly-used substances that inhibit or inactivate RNases, including detergents, chaotropes, organics, metals, proteases and metals are inappropriate for use in target amplification systems because they also inhibit the enzymes used for amplification. RNase-inhibiting proteins, e.g. human placental RNase inhibitor (Blackburn et al., 1977, J. Biol. Chem. 252: 5904) or rat liver RNase inhibitor (Gribnau et al., 1969, Arch. Biochem. Biophys. 130: 48-52), may be unstable, are expensive, and can contribute additional interfering substances, such as nucleic acids and RNases that are not inhibited by the inhibitor.
In addition to nucleases, traces of other enzymes, nucleic acids, and certain buffer salts may interfere with amplification reactions. While these substances are merely undesirable for many uses of RT, because of the nature of the amplification reaction, it is critical that RT preparation contain as little contaminating substances as possible.
Reverse transcriptases have been isolated and purified from various sources. When RT is isolated directly from virus particles, cells or tissues, high costs may preclude their widespread use in diagnostic tests (e.g., see Kacian et al., 1971, Biochim. Biophys. Acta 46: 365-83; Yang et al., 1972, Biochem. Biophys. Res. Comm. 47: 505-11; Gerard, et al., 1975, J. Virol. 15: 785-97; Liu et al., 1977, Arch. Virol. 55 187-200; Kato et al., 1984, J. Virol. Methods 9: 325-39; Luke et al., 1990, Biochem. 29: 1764-69 and Le Grice et al., 1991, J. Virol. 65: 7004-07). Also, these methods have not assured removal of inhibitors or contaminants that interfere with target amplification reactions. Another important consideration for a variety of reverse transcriptase uses is the RT-associated RNase H activity. The amount of RNase H activity and coordination of RNase H activity with the RNA- and DNA-dependent RT activities are important features that affect an enzyme""s utility for various purposes, including transcription-based amplification systems. Too much or too little activity, inappropriate activity (e.g., non-specific RNase activity), or poorly-coordinated RNase H and DNA synthesis activities can all lead to reduced performance. Proper balance of the synthetic and degradative activities must be maintained; this is not only a function of the particular RT used, but also depends on the ability of a purification protocol to remove inappropriate RNase and/or DNase activities.
Reverse transcriptase genes have been cloned and expressed in bacterial hosts. Attempts to clone and express in E. coli a gene encoding reverse transcriptase from avian myeloblastosis virus (AMV-RT) did not lead to production of significant amounts of purified enzyme. This is probably because AMV-RT consists of two polypeptide chains (xcex1 and xcex2) which must form a dimer and undergo specific post-translational modifications to produce a fully active enzyme. These modifications do not occur in E. coli. 
In contrast to AMV-RT, many reverse transcriptases derived from mammalian viruses consist of only one polypeptide chain; cloning and expression of these enzymes have been more successful. Goff et al. (U.S. Pat. No. 4,943,531) and Kotewicz et al. (U.S. Pat. No. 5,017,492) have described methods for the purification of reverse transcriptase derived from Moloney Murine Leukemia Virus (MMLV-RT) and expressed in E. coli. These methods form the basis of many commercially available RT.
Some protein purification methods use affinity tags attached to the protein of interest which is used to select the protein of interest from a mixture by binding the affinity tag to its ligand. Affinity tags include, for example, histidine residues, glutathione S-transferase, Protein A or maltose binding protein.
Many commercial RT preparations have been found unsuitable for use in target amplification and for other purposes due to nuclease contamination (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ryskov et al.,1982, Mol. Biol. Rep. 8: 213-16). Other problems with commercial MMLV-RT preparations may be related to an altered coordination between the DNA synthesis and RNAse H activities of the purified enzyme, reduced ability to bind and initiate synthesis at primer sites or to read through regions of tight secondary structure, or contaminating DNase and other proteins (Agronovsky A. A., 1992, Anal. Biochem. 203: 163-65). Also, commercial preparations made using the previously available purification methods show significant lot-to-lot variability. Moreover, due in part to lengthy and labor-intensive purification procedures, the expense of the reagents and scale-up equipment, and the low enzyme yields, the cost is prohibitive for widespread commercial application of the enzymes in target amplification systems.
It is therefore an object of the present invention to provide an improved form of reverse transcriptase having the correct balance of DNA synthetic activities and RNAse H digestive activities, thereby being particularly suited for use in nucleic acid amplification methods.
It is another object of the present invention to provide a convenient source of reverse transcriptase containing low levels of contaminants (e.g., undesired RNases) that interfere with transcription-based amplification reactions by cloning and expressing a gene encoding MMLV-RT having these properties in an E. coli host.
It is another object of the present invention to reduce the RNase activity associated with the enzyme prior to and following purification by cloning and expressing the MMLV-RT gene in a ribonuclease-deficient strain of E. coli. 
It is another object of the present invention to develop a simple purification scheme for the isolation of the RT enzyme.
It is also an object of the present invention to provide methods for the purification of the enzyme that achieve high levels of RT purity at a low cost.
The present invention features an expression vector or plasmid containing a cloned version of the gene for MMLV-RT which, when used to transform a suitable host cell such as E. coli, leads to expression of the gene and generation of a gene product having the DNA- and RNA-directed DNA polymerase activities and RNAse H activity associated with retroviral reverse transcriptases.
The present invention also features a plasmid containing a MMLV-RT gene inserted into a host cell which has a reduced level of ribonuclease activity as compared to wild-type strains.
The present invention also includes methods for the purification of the resulting enzyme from the host cells, such methods comprising suitable growth media, fermentation conditions, harvesting and storage of the cells, cell lysis and chromatography.
The present invention also features the enzyme produced by the expression vectors, host cells, and purification procedures of the present invention. The enzyme is highly-purified and suitable for use in nucleic acid amplification and other genetic engineering procedures.
The present invention features the use of the enzyme produced by the methods described herein for the synthesis of cDNA for a variety of purposes, notably in transcription-based amplification and RT-PCR reactions.
According to one aspect of the invention, there is provided a recombinant DNA molecule that includes a DNA fragment containing a DNA sequence encoding a single-chain polypeptide derived from Moloney murine leukemia virus (MMLV) having RNA-directed and DNA-directed DNA polymerase activities and RNase H activity and encoding a plurality of contiguous histidine residues in then single-chain polypeptide; a DNA fragment comprising a promoter sequence for expressing the gene encoding the single-chain polypeptide in an E. coli host cell; and a DNA fragment containing an origin of replication that promotes autonomous replication of a vector in an E. coli host cell. In the recombinant DNA molecule, the DNA fragments are operably linked so that the fragments are replicated together in the E. coli host cell and the DNA sequence encoding the single-chain polypeptide is expressed in the E. coli host cell to produce the single chain polypeptide. In one embodiment, the DNA sequence encoding the plurality of contiguous histidine residues is located at or near the 5xe2x80x2 or 3xe2x80x2 end of the DNA sequence encoding the single-chain polypeptide derived from MMLV. In another embodiment, the DNA sequence encoding the plurality of contiguous histidine residues encodes six histidine residues. The DNA sequence encoding the plurality of contiguous histidine residues may be located adjacent to a codon encoding an amino terminal glycine residue of the single-chain polypeptide derived from MMLV, or may be located adjacent to a codon encoding a carboxyl-terminal stop signal for expression of the single-chain polypeptide derived from MMLV in the E. coli host cell.
According to another aspect of the invention, there is provided a method for producing a polypeptide having RNA-directed and DNA-directed DNA polymerase activities. The method includes the steps of providing a plasmid comprising a DNA sequence derived from a Moloney murine leukemia virus (MMLV) sequence and encoding a single-chain polypeptide having RNA-directed and DNA-directed DNA polymerase activities and RNase H activity, codons encoding a plurality of contiguous histidine residues located at or near either an amino-terminus or carboxyl-terminus of the single-chain polypeptide, at least one selectable marker gene, a promoter sequence for expression of the DNA sequence derived from MMLV in an E. coli host cell, and an origin of replication for autonomous replication of the plasmid within an E. coli host cell and growing E. coli host cells containing the plasmid in a liquid culture that promotes cell division and expression of the DNA sequence derived from MMLV. Then, the method includes lysing the E. coli host cells to form a cell lysate; and purifying the single-chain polypeptide from the cell lysate using metal ion affinity chromatography that uses the contiguous histidine residues present in the single-chain polypeptide. In one embodiment, the metal ion affinity chromatography is performed using nickel ions attached to a resin to retain a His-tagged reverse transcriptase enzyme derived from the MMLV sequence. In another embodiment, the His-tagged reverse transcriptase enzyme is eluted from the nickel ions attached to the resin using an imidazole-containing buffer. The single-chain polypeptide purified using metal ion affinity chromatography preferably has an apparent molecular weight of about 70,000 daltons. The single-chain polypeptide purified using metal ion affinity chromatography preferably has DNA-directed DNA polymerase activity having a specific activity of at least about 275 U/mg as determined using a primer extension reaction and by comparison to DNA-directed DNA polymerase activity of a known reverse transcriptase enzyme.